Many chromosomal abnormalities and genetic instability arising in cancer and other human diseases are caused by defects in chromosomal replication. Chromosomal replication not only requires the proper function of the basic replication machinery, but also demands an ability to cope with the complex nature of chromatin and lesions on the template, both of which can lead to replication fork stalling or collapse. Checkpoint pathways have been shown to play key roles in mediating multiple measures that promote the recovery and completion of replication. In addition, special mechanisms that can coordinate chromosomal behavior and basic DNA replication programs are critical. SMC (structural maintenance of chromosome) proteins and their complexes, including the newly characterized Smc5/6 complex, are dynamic molecular linkers that can actively tether and fold chromatids, thereby providing important connections between chromosomal structures and replication programs. Our long-term goal is to understand the special programs utilized in chromosomal replication. In this grant period, we will focus on functions of the Smc5/6 complex in replication. We showed that the Smc5/6 complex plays multiple roles in chromosomal replication and some of its roles are mediated by its SUMO E3 subunit Mms21. Using a combination of genetic, cell biological, and biochemical approaches, we will study the mechanisms by which the Smc5/6 complex regulates chromosomal replication. In specific aim one, we will determine how the Smc5/6 complex counteracts recombinogenic events at damaged replication forks. In specific aim two, we will study the additional functions of the Smc5/6 complex in replication and probe for physical interactions between the Smc5/6 complex and proteins important for replication. The combined results from these two aims will provide new insights into the relationship between replication, chromosomal dynamics and recombinational repair. It will likely contribute to the understanding of the functions of the human Smc5/6 complex and potentially lead to methods to battle genomic instability associated with cancer and other human diseases. Chromosomal replication faces many challenges posed by the complex nature of chromatin and nuclear organization. It therefore requires special mechanisms that coordinate chromosomal behavior and basic DNA replication and repair programs. The evolutionarily conserved Smc5/6 complex functions at the interface of these processes. We describe research programs to address the mechanisms by which this complex co- regulates different pathways during chromosomal replication. Results from these studies will provide new insights into these fundamental processes and will shed lights on the cause and treatment of human diseases related to defects in these processes.